Method of immunoassay tartrate resistant acid phosphatase 5b and kit to be used therein

ABSTRACT

Specific and accurate immunoassay of tartrate resistant acid phosphatase 5b (TRACP 5b) in a specimen may be carried out by bonding TRACP 5b in the specimen to an antibody, subjecting the TRACP 5b bonded to the antibody to enzyme reaction with a 2-halo-4-nitrophenylphosphoric acid or a salt thereof, a substrate for TRACP 5b, and then assaying the enzymatic activity of TRACP 5b.

TECHNICAL FIELD

The present invention relates to a method for immunoassay of tartrateresistant acid phosphatase activity derived from osteoclast as a boneresorption marker and a kit to be used therein. According to the presentinvention, specific assay of tartrate resistant acid phosphataseactivity derived from osteoclast is possible and this activity is veryeffective as a bone resorption marker in the fields of medicaltreatments and clinical examinations of bone diseases.

BACKGROUND ART

A large portion of tartrate resistant acid phosphatase (TRACP) in serumis considered acid phosphatase derived from osteoclast and the assay ofTRACP is considered useful as an indication for evaluating the functionof osteoclast. Thus, TRACP is gaining interest as a bone resorptionmarker (Norio Fukunaga, Toshitaka Nakamura and Toshio Matsumoto,“Osseous Metabolism Marker”, Medical Review Co., Ltd., 1995). On theother hand, acid phosphatase in serum is divided into six bands 0 to 5from the origin by polyacrylamide gel electrophoresis. Of these, acidphosphatase corresponding to the fifth band is tartrate-resistant and iscalled Band 5 tartrate resistant acid phosphatase (TRACP 5: tartrateresistant acid phosphatase 5). This acid phosphatase is further dividedby electrophoresis into 5a which has a high content of sialic acidbonded to a sugar chain and 5b which has almost no sialic acid bonded toa sugar chain. In addition, 5a is an enzyme derived from cells otherthan osteoclast and its blood level does not vary, while only the bloodlevel of 5b varies with bone resorption. Therefore, it is consideredthat 5b is the main substance of tartrate resistant acid phosphatasederived from osteoclast. Also in “Clinical Chemistry” (Clin. Chem. 47:1497. 2001), it is recommended that ACP derived from osteoclast shouldbe abbreviated as TRACP 5b. Accordingly, also in the presentspecification, phosphatase that refers to ACP derived from osteoclastand used as an indication of bone resorption is expressed in the term“TRACP 5b”, and tartrate resistant acid phosphatase derived fromosteoclast and tartrate resistant acid phosphatase 5b are hereinafterconsidered synonymous with each other. Thus, all of them are hereinafterexpressed in the term “TRACP 5b” in the present specification.

Conventional activity assay methods in which TRACP activity is assayedas an indication of acid phosphatase capable of indicating the activityof osteoclast are disadvantageous in specificity, sensitivity,troublesomeness of assay and assay time.

In general, in the assay of TRACP 5b by the activity assay method, theenzymatic activity is assayed by colorimetric determination of areaction product (an alcohol or a phenol) produced by an enzymereaction, by using a phosphoric ester as a synthetic substrate in thepresence of tartaric acid. In this case, tartaric acid inhibits acidphosphatase derived from prostate and the residual acid phosphataseactivity is assayed with the substrate to assay TRACP activity as TRACP5b activity. However, besides tartrate resistant acid phosphatasederived from osteoclast, that derived from erythrocyte or that derivedfrom platelet is present in a specimen, and such acid phosphatase isalso assayed. Therefore, the above TRACP 5b assay method isdisadvantageous in specificity.

As a modification of such a method, there is known a method in whichafter a pretreatment comprising incubation of a 5-fold dilution of serumat 37° C. for 1 hour, the residual TRACP activity is assayed by the useof p-nitrophenylphosphoric acid as a substrate in the presence oftartaric acid (“Nichidai-Ishi”, 49:904-911. 1990; and Clin. Chem.33:458-462. 1987). This method permits avoidance of the influence ofacid phosphatase derived from erythrocyte but does not permit exclusionof the influence of acid phosphatase derived from platelet. In addition,the present inventors have reported a TRACP 5b assay method utilizingthe difference in sensitivity to fluorine between TRACP 5b and tartrateresistant acid phosphatase activity derived from erythrocyte orplatelet, as a more specific activity assay method (JP-A-10-37198). Thismethod, however, does not permit exclusion of the influence of TRACP 5athough it permits avoidance of the influence of tartrate resistant acidphosphatase derived from erythrocyte or platelet. Moreover, this methodis disadvantageous in precision because TRACP 5b activity is assayed inthis method by subtracting activity not inhibited in the presence offluorine from the total tartrate resistant acid phosphatase activity. Inaddition, a method has been reported in which TRACP 5b activity isassayed by using an inhibitor for TRACP 5a in combination with theabove-mentioned method utilizing fluorine (JP-A-2001-231595). However,since TRACP 5b activity is assayed by the subtraction also in thismethod, this method is disadvantageous in precision like the methodusing only fluorine, though it is more specific than the latter method.

On the other hand, as TRACP 5b assay methods using immunoassay, thereare also known immunoassay methods using a polyclonal antibody or amonoclonal antibody (J Clin Endocrinol Metab. 71:442-451. 1990; J BoneMiner Res. 13:683-687. 1998; Immunol Lett. 70:143-149. 1999; J BoneMiner Res. 14:464-469. 1999; Clin Chem. 45:2150-2157. 1999; and ClinChem. 46:1751-1754. 2000). In these methods, activity corresponding tothe whole of Band 5 is assayed, so that the influence of TRACP 5a is notnegligible. In addition, an immunoassay method has been reported inwhich TRACP 5b is more specifically assayed (Japanese Patent ApplicationKohyo No. 2002-510050). It has been reported that this method is anassay method more specific for TRACP 5b activity because in this method,measured values are obtained by activity assay by utilizing thedifference in optimum pH between TRACP 5a and TRACP 5b. However, sincethe antibody used in this method is not specific for TRACP 5b, thismethod is not always sufficient in specificity for TRACP 5b and hence isdesired to be further improved in order to use TRACP 5b as a boneresorption marker.

DISCLOSURE OF THE INVENTION

In view of such problems, the present invention is intended to provide amethod for assaying TRACP 5b as a bone resorption marker specificallywith high sensitivity, and a kit to be used in this method.

The present invention provides a method for immunoassay of tartrateresistant acid phosphatase 5b (TRACP 5b) in a specimen which comprises

bonding TRACP 5b in the specimen to an antibody, subjecting the TRACP 5bbonded to the antibody to enzyme reaction with a2-halo-4-nitrophenylphosphoric acid represented by the general formula(1):

wherein X is Cl, F, Br or I, or a salt thereof, which is a substrate forTRACP 5b, and then assaying the enzymatic activity of TRACP 5b to carryout the immunoassay of TRACP 5b in the specimen.

The present invention also provides a kit for immunoassay of TRACP 5bwhich comprises

i) a solid support,

ii) an antibody against TRACP 5b, and

iii) a substrate for TRACP 5b which is a 2-halo-4-nitrophenylphosphoricacid represented by the general formula (1):

wherein X is Cl, F, Br or I, or a salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the optimum pH in the assay of the enzymaticactivity of each of TRACP 5b and TRACP 5a by the use of2-chloro-4-nitrophenylphosphoric acid (CNPP) as a substrate for enzyme.

FIG. 2 is a graph obtained by treating a specimen (a standard solutionor serum) with a plate having an antibody against TRACP 5b immobilizedthereon, and then assaying the enzymatic activity by a rate method byusing 2-chloro-4-nitrophenylphosphoric acid as a substrate for enzyme.

MODE FOR CARRYING OUT THE INVENTION

The specimen to be subjected to assay in the present invention includeshuman blood, serum, plasma and the like and is not particularly limitedso long as it is likely to contain TRACP 5b.

In the present invention, the antibody is preferably bonded to a solidsupport. In addition, as the antibody, either a monoclonal antibody or apolyclonal antibody may be used so long as they are antibodies againstTRACP 5b, though the monoclonal antibody is preferable from theviewpoint of specificity. As the monoclonal antibody, heretofore-knownmonoclonal antibodies against TRACP 5b may be used.

As the antibody used in the present invention, a monoclonal antibodyagainst TRACP 5b may be prepared by using TRACP 5b purified from humanosteoclast, as an immunogen. The monoclonal antibody is produced, forexample, by hybridoma obtained by immunizing an animal with purifiedhuman TRACP 5b as an immunogen and fusing cells capable of producinganti-human TRACP 5b antibody, which are produced by the animal, withmyeloma cells.

The above-mentioned hybridoma may be obtained by the following method.That is, human TRACP 5b is mixed with a well-known adjuvant such asFreund's complete or incomplete adjuvant, aluminum hydroxide adjuvant,pertussis adjuvant or the like to prepare an adjuvant liquid forsensitization, and this liquid is administered to an animal (e.g. amouse or a rat) subcutaneously in the abdominal cavity or intravenouslyin the tail, in several portions at intervals of 1 to 3 weeks toimmunize the animal. Although the amount of the antigen used for thesensitization is usually chosen in the range of 1 μg to 100 mg, it ispreferably about 50 μg in general. Although the number of immunizingoperations is generally 2 to 7, various methods are known. Subsequently,antibody-producing cells derived from the spleen or the like are fusedwith cells having proliferating capability in a test tube, such asmyeloma cells or the like. The antibody-producing cells may be obtainedfrom the spleen or the like of a mouse, a nude mouse, a rat or the like.

As to a method for the fusion, the fusion may be carried out by the useof a poly(ethylene glycol) (PEG) by the method of Köhller and Milstein(Nature. 256, 495. 1975) which is already per se well known. The fusionmay be carried out also by the use of Sendai virus or by anelectrofusion method.

As to a method for selecting hybridoma capable of producing an antibodycapable of recognizing human TRACP 5b, from the fused cells, theselection may be carried out as follows. That is, the hybridoma isselected from colonies formed by cells surviving in HAT medium and HTmedium in limiting dilution of the fused cells. When an antibody againsthuman TRACP 5b is contained in the supernatant of the culture medium forany of the colonies formed by the fused cells seeded into a 96-wellplate or the like, a clone capable of producing a monoclonal antibodyagainst human TRACP 5b may be selected by an ELISA method in which thesupernatant is placed on an assay plate having human TRACP 5bimmobilized thereon, and after the reaction, a secondary labeledantibody such as an anti-mouse immunoglobulin-HRP labeled antibody isreacted with the above-mentioned antibody. As the labeling substance ofthe labeled antibody, there may be used enzymes (e.g. alkalinephosphatase), fluorescent substances, radioactive substances and thelike besides HRP. Screening of specific antibodies against human TRACP5b may be conducted by carrying out, as a control, ELISA using an assayplate having only BSA bonded thereto as a blocking agent, simultaneouslywith the above-mentioned ELISA. That is, a clone may be selected whichis positive on the plate having human TRACP 5b immobilized thereon andis negative in the ELISA using only BSA.

As the thus selected hybridomas capable of producing a monoclonalantibody against TRACP 5b, hybridomas TrK27, TrK49 and TrK62 may beexemplified. Hybridomas TrK27, TrK49 and TrK62 were deposited as followsin Patented Organism Deposition Center (IPOD), Industrial TechnologyGeneral Research Institute (Independent Administrative Corporation),Chuo-dairoku, Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture, Japan305-8566: hybridoma TrK27 was deposited as a receipt number IPODFERMBP-7889 on Feb. 14, 2002, hybridoma TrK49 was deposited as a receiptnumber IPOD FERM BP-8249 on Nov. 27, 2002, and hybridoma TrK62 wasdeposited as a receipt number IPOD FERMBP-7890 on Feb. 14, 2002.

Each of the hybridomas is cultured on a medium usually used for cellculture, such as α-MEM, RPMI1640, ASF, S-clone or the like, and themonoclonal antibody may be recovered from the supernatant of the culturemedium. The following is also possible: after an animal from which thehybridoma has been derived, such as a mouse is previously treated withpristine, the hybridoma is intraperitoneally injected into the animal tocause accumulation of ascites, and the monoclonal antibody is recoveredfrom the ascites. As a method for recovering the monoclonal antibodyfrom the supernatant or the ascites, a conventional method may beadopted. There are exemplified salting-out with ammonium sulfate, sodiumsulfate of the like, chromatography, ion exchange chromatography, andaffinity chromatography using protein A, protein G or the like.

As the monoclonal antibody used in the present invention, a monoclonalantibody reactive with not only TRACP 5b but also TRACP 5a may be used.In the present invention, a polyclonal antibody may be used. Thepolyclonal antibody may be obtained, for example, by using TRACP 5bpurified from human osteoclast, as an immunogen, immunizing an animalsuch as a rat or mouse with this TRACP 5b, and preparing antiserum fromthe animal.

In the present invention, the immunoassay of TRACP 5b in a specimen iscarried out by bonding TRACP 5b in the specimen to the above-mentionedantibody, subjecting the bonded TRACP 5b to enzyme reaction with a2-halo-4-nitrophenylphosphoric acid of the general formula (1) or a saltthereof, as a substrate for enzyme, and then assaying the enzymaticactivity.

As the 2-halo-4-nitrophenylphosphoric acid used in the presentinvention, there may be exemplified 2-chloro-4-nitrophenylphosphoricacid, 2-fluoro-4-nitrophenylphosphoric acid and2-bromo-4-nitrophenylphosphoric acid. As the salt of the2-halo-4-nitrophenylphosphoric acid, there may be exemplified itsammonium salt, imidazolium salt, cyclohexylammonium salt, potassiumsalt, sodium salt and tris(hydroxyammonium) salt.

The above-mentioned substrate used in the present invention has a lowerKm value for TRACP 5b and a higher Km value for TRACP 5a than doesp-nitrophenylphosphoric acid (PNPP), and hence is very advantageous insensitivity and specificity when used for assaying TRACP 5b.

In the present invention, when TRACP 5b in a specimen is subjected toenzyme reaction with the 2-halo-4-nitrophenylphosphoric acid or itssalt, a substrate for TRACP 5b, the pH at the reaction is preferablyhigher than 6.3 and not higher than 6.8, more preferably 6.35 to 6.75,in particular, 6.38 to 6.70. When the pH is too low, the enzyme reactionof TRACP 5a becomes liable to occur, resulting in a low ratio of TRACP5b reactivity to TRACP 5a reactivity. Therefore, the specificity forTRACP 5b tends to be insufficient. When the pH is too high, the enzymereaction of TRACP 5b itself does not occur easily, so that themeasurement sensitivity tends to be lowered.

In the present invention, specifically, TRACP 5b may be assayed asfollows by adopting, for example, an end-point method. At first, aspecimen to be subjected to assay is added to an antibody adsorbed on asolid support to subject TRACP 5b in the specimen and the antibody toantigen-antibody reaction and bond TRACP 5b to the antibody. Then, thesolid support is washed with a washing solution to remove componentscontained in the specimen and not adsorbed on the antibody. Thereafter,the 2-halo-4-nitrophenylphosphoric acid or its salt is added to thereaction system as a substrate for enzyme to react the substrate withthe TRACP 5b bonded to the antibody, preferably at a pH of higher than6.3 and not higher than 6.8. After the enzyme reaction is terminatedwith a reaction-terminating solution, the 2-halo-4-nitrophenol producedby the reaction is subjected to absorbance measurement at a wavelengthof 390 nm to 450 nm, preferably 400 to 430 nm. Since the value of theabsorbance reflects the enzymatic activity of TRACP 5b, TRACP 5b in thespecimen may be assayed on the basis of the absorbance value.

In the present invention, rate assay may also be carried out as theassay of the enzymatic activity unlike in a conventional method usingp-nitrophenylphosphoric acid because the 2-halo-4-nitrophenol develops acolor at the pH at the reaction. When the rate assay is carried out,TRACP 5b in a specimen may be assayed by measuring the reactivity ofTRACP 5b with the substrate as an average absorbance change per adefinite time (usually 1 minute). As a result, the measurement time isreduced, which is more desirable.

In the present invention, as is clear from the assay method describedabove, the antibody is preferably used after being bonded to a solidsupport. The solid support is not particularly limited though a solidsupport used in a solid phase immunoassay method such as ELISA isusually used. A material for solid support includes, for example,polystyrenes, polypropylenes, polycarbonates, polyethylenes, nylons andmethacrylates. As the shape of the solid support, a plate shape and abead shape are exemplified.

For preparing the antibody adsorbed on the solid support, the antibodyagainst TRACP 5b is directly or indirectly bonded to the solid supportby utilizing physical bonding, chemical bonding or affinity. The amountof the antibody used for the sensitization is often in the range of 1 ngto 100 mg/ml.

The method of the present invention may be practiced by the use of a kitfor immunoassay of TRACP 5b which comprises i) a solid support, ii) anantibody against TRACP 5b, and iii) a substrate that is a2-halo-4-nitrophenylphosphoric acid of the general formula (1) or a saltthereof.

In this kit, as to i) the solid support and ii) the antibody againstTRACP 5b, it is possible to prepare the solid support and a solution ofthe antibody separately and adsorb the antibody on the solid support atthe time of assay of TRACP 5b. Alternatively, the kit may be providedafter the previous adsorption of the antibody on the solid support. Thekit preferably comprises a washing solution for the purpose of removingcomponents not adsorbed on the solid support, after the bonding of TRACP5b in a specimen to the antibody. For example, Tris buffer containing asurfactant may be used as the washing solution.

When this kit is used in an end-point method, the kit preferablycomprises an enzyme-reaction-terminating solution. As theenzyme-reaction-terminating solution, there may be used, for example,aqueous alkali solutions such as solutions of potassium hydroxide,sodium hydroxide and the like.

In addition, the kit of the present invention may further comprises adiluent for specimen if necessary. For example, a buffer solution suchas Tris buffer may be used as the diluent for specimen. If necessary, achelating agent (e.g. EDTA.2Na) and an inorganic salt (e.g. sodiumchloride) may be added to the buffer solution.

The present invention is illustrated in further detail with reference tothe following reference examples and working examples, which should notbe construed as limiting the scope of the invention.

REFERENCE EXAMPLE 1

Preparation of a Monoclonal Antibody Against TRACP 5b

(1) Purification of Acid Phosphatase TRACP 5b Derived from Osteoclast

After informed consent was obtained, 130 g of the caput of human thighbone excised by a surgical operation was frozen in liquid nitrogen,crushed with a hammer, and then suspended in 200 mL of a buffer solution(50 mM Tris-HCl, 0.3M KCl, 1 mM PMSF, 1 mM EDTA.2Na, 0.1% Triton X-100,0.02% NaN₃, 1 unit/ml aprotinin, pH 7.5) containing a proteaseinhibitor, followed by homogenization in a ultrasonic homogenizer. Thehomogenate was stirred overnight at 4° C. and centrifuged at 10,000 rpmfor 20 minutes, and the supernatant was dialyzed against 10 mM Trisbuffer (pH 8.2). Thereafter, the dialyzed solution was applied to aCM-Sepharose column (Φ40 mm×40 cm) [Sigma Chemical Co.] and the proteinadsorbed was eluted with the same Tris buffer as above containing NaCl,while increasing the NaCl concentration with a linear concentrationgradient (0-0.5M NaCl). Tartrate resistant acid phosphatase activity wasassayed with a substrate 2,6-dichloro-4-acetylphenylphosphoric acid[mfd. by Nitto Boseki Co., Ltd.], and fractions containing a high levelof the activity were pooled. The solution thus obtained was concentratedand then dialyzed against 20 mM Tris buffer (pH 7.2) containing 0.7 MNaCl, and the dialyzed solution was applied to a Superdex 200 column(Φ16 mm×60 cm) [Amersham Pharmacia Biotech AB]. In the same manner asabove, tartrate resistant acid phosphatase activity in fractionsobtained by elution was assayed and fractions containing the activitywere pooled. The solution thus obtained was diluted 2-fold with 20 mMTris buffer (pH 7.2) and applied to a HiTrap Heparin HP column (5 mL)[Amersham Pharmacia Biotech AB], and the protein adsorbed was elutedwith the same 20 mM Tris buffer (pH 7.4) as above containing NaCl, whileincreasing the NaCl concentration with a linear concentration gradient(0.35-1M NaCl). Fractions containing a high tartrate resistant acidphosphatase activity were pooled and then concentrated to obtain 0.4 mgof purified acid phosphatase derived from osteoclast. The amount of theprotein was confirmed by A₂₈₀. As to the purity, SDS-PAGE [TIFCO] wascarried out, followed by silver staining. As a result, the purity wasconfirmed by the appearance of a single band at a molecular weight ofabout 35,000. The enzyme corresponding to the single band was consideredas purified TRACP 5b and was used as an immunizing antigen.

(2) Immunization

The purified tartrate resistant acid phosphatase derived from humanosteoclast (TRACP 5b) was diluted to a concentration of 250 μg/ml with50 mM citrate buffer (pH 5.5), and 25 μg (100 μl) of the dilution wasthoroughly mixed with 100 μl of Freund's complete adjuvant [Wako PureChemical Industries, Ltd.] until emulsification was effected. Thesuspension thus prepared was intraperitoneally administered to a Balb/cfemale mouse aged 6 weeks [Nippon Clear Co., Ltd.] under anesthesia withdiethyl ether. After 2 weeks, the same amount as above of TRACP 5b (25μg/ml) was mixed with Freund's incomplete adjuvant [Wako Pure ChemicalIndustries, Ltd.]. By exactly the same operation as in the case of theFreund's complete adjuvant, emulsification was effected to obtain asuspension and the mouse was sensitized with the suspension. Two weeksafter this procedure, the same procedure as above was carried out. Forthe fourth immunization, i.e., final immunization, a dilution of TRACP5b (25 μg/ml) with 50 mM citrate buffer (pH 5.5) was prepared and thenadministered to the mouse by injection into the tail vein.

(3) Establishment of Hybridoma

Three days after the final immunization, the spleen was surgicallyremoved from the mouse sensitized with TRACP 5b, under anesthesia withdiethyl ether, and was aseptically dispersed to prepare splenocytes.Fusion was carried out according to the method of KÖhller and Milstein(Nature, 256, 495, 1975). The splenocytes were fused with myeloma cellsP3-X63-Ag8-U1 (P3U1) by the use of a poly(ethylene glycol) (PEG4000)[Merck & Co., Inc.]. As to the fusion ratio, the number of thesplenocytes was 8×10⁷, while the number of the myeloma cellsP3-X63-Ag8-U1 (P3U1) was 2×10⁷. That is, the fusion ratio of thesplenocytes to the myeloma cells was 4:1. The fused cells were dispersedin 10% FCS [INVITROGEN] α-MEM [IRVINE] HAT [Cosmo Bio Co., Ltd.] medium,seeded into a 96-wells microtiter culture plate [Sumitomo Bakelite Co.,Ltd.] and then cultured under conditions of 37° C. and 5% CO₂.

(4) Screening

After about 2 weeks, the growth of colonies was confirmed and screeningwas conducted. A method for conducting the screening is described below.For producing a plate for the screening, TRACP 5b purified in the aboveitem (1) was dissolved in 50 mM citrate buffer and applied to a 96-wellplate [Nunc] in an amount of 0.5 μg/100 μl/well. The plate was allowedto stand at 4° C. for two nights and then washed three times with Trisbuffer containing 0.05% Tween 20. To each well was applied 200 μl of1.5% BSA solution in order to inhibit a nonspecific reaction, and theplate was allowed to stand overnight at 4° C. After the thus completedplate was washed three times with Tris buffer containing 0.05% Tween 20,100 μl of the culture supernatant was reacted in each well and the platewas further washed. Then, HRP-labeled anti-mouse immunoglobulin antibody[Zymed Laboratories, Inc.], a secondary antibody was added to carry outthe reaction. After washing, 100 μl of a 3 mg/ml color-producingsolution of o-phenylenediamine (OPD) [Nacalai Tesque Inc.], acolor-producing substrate for HRP, in citric acid was added to each wellto cause coloration for a definite period. Then, 100 μl of 1N sulfuricacid was added to each well as a terminating solution and absorbance wasmeasured at a measuring wavelength of 492 nm. Clones found to bepositive by the above procedure were subjected to recloning by alimiting dilution method, and the supernatants thus obtained werechecked again.

(5) Confirmation of an Antibody

The reaction of clone TrK27 with purified TRACP 5b was confirmed byELISA and clone TrK27 was selected as a clone capable of producing anantibody reactive with TRACP 5b. This hybridoma TrK27 was deposited as areceipt number IPOD FERMBP-7889 on Feb. 14, 2002 in Patented OrganismDeposition Center (IPOD), Industrial Technology General ResearchInstitute (Independent Administrative Corporation), Chuo-dairoku,Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture, Japan 305-8566.

An antibody produced by this hybridoma was assayed by the use of amonoclonal antibody typing kit [Amersham Pharmacia Biotech AB] to findthat its class was IgG1 and that its light chain was K. The specificityof this antibody was investigated as follows. ELISA was carried out byusing, as a specimen, each of TRACP 5b, TRACP 5a derived from humanserum and eluted from a HiTrap Heparin HP column, ACP derived fromprostate, ACP derived from platelet (a platelet extract solution) andACP derived from erythrocyte (an erythrocyte extract solution). As aresult, the antibody reacted with TRACP 5b and TRACP 5a but not with theother isoforms.

(6) Preparation and Purification of a Monoclonal Antibody

To a Balb/c female mouse aged 10 weeks [Nippon Clear Co., Ltd.] twoweeks after administration of 0.5 ml of pristane [Aldrich Chemical Co.]to the mouse were intraperitoneally administered 1×10⁷ cells of theobtained hybridoma TrK27. After about 2 weeks, ascites accumulated inthe abdominal cavity of the mouse was surgically collected underanesthesia with diethyl ether. As a result of confirmation by the use ofthe ascites stepwise diluted as a sample and by the ELISA method adoptedfor the screening, it was found that the ascites contained a highconcentration of a monoclonal antibody. The ascites was treated with 40%ammonium sulfate and dialyzed against PBS, and then the monoclonalantibody was purified by the use of a protein G column [AmershamPharmacia Biotech AB] and confirmed by SDS-PAGE. As a result, a singleband was confirmed at a molecular weight of about 150,000 when theantibody was in a non-reduced state, and two bands were confirmed atmolecular weights of about 50,000 and 25,000 when the antibody had beenreduced with mercaptoethanol. The amount of the purified antibody wasabout 15 mg per mouse, namely, it was sufficient for industrialutilization.

(7) Production of a Plate having an Anti-TRACP 5b Antibody ImmobilizedThereon

The antibody obtained in the above item (6) was dissolved in PBS and theresulting solution was applied to a 96-well plate [Nunc] in an amount of1 μg/200 μl/well. The plate was allowed to stand at 4° C. for two nightsand then washed three times with Tris buffer containing 0.05% Tween 20.To each well was applied 200 μl of 1.5% BSA solution in order to inhibita nonspecific reaction, and the plate was allowed to stand overnight at4° C. to complete a plate having an anti-TRACP 5b antibody immobilizedthereon.

REFERENCE EXAMPLE 2

Comparison Between the Substrate Used in the Present Invention andAnother Substrate by Km Measurement

In order to investigate the difference between TRACP 5b and TRACP 5a inreactivity with 2-chloro-4-nitrophenylphosphoric acid (CNPP), Km foreach of them was measured. As a result, it was found that Km was 1.5 mM(pH 6.4) for TRACP 5b and 3.5 mM (pH 5.6) for TRACP 5a. The Km value forTRACP 5b is less than one-half that for TRACP 5a, indicating that thesubstrate CNPP is more suitable for the assay of TRACP 5b than for thatof TRACP 5a.

On the other hand, according to Clin. Chem., 47(1), 74-80, Km values inthe reaction of each of TRACP 5b and TRACP 5a withp-nitrophenylphosphoric acid (PNPP) are 7.0 mM (pH 5.8) and 1.9 mM (pH5.2), respectively. That is, for the assay of TRACP 5b, CNPP used in thepresent invention is more advantageous than PNPP from the viewpoint ofaffinity and specificity.

EXAMPLE 1

Assay of TRACP 5b or 5a at Various pH Values

The pH of a buffer solution containing a substrate CNPP was varied from5.6 to 6.8 and the activity of TRACP 5b and TRACP 5a for this substratewas assayed. This substrate buffer solution had the followingcomposition: CNPP 5 mM, Mes 0.1 M and sodium tartrate 40 mM. Its pH wasadjusted to values varying from 5.6 to 6.8 by steps of 0.2. TRACP 5b andTRACP 5a used as samples were prepared by separating human serum by theuse of a Heparin column and concentrating a fraction corresponding to apeak due to each of TRACP 5a and TRACP 5b. As TRACP 5a, there was used asample having such a concentration that its absorbance was 0.8 at pH 5.6(the optimum for TRACP 5a). As TRACP 5b, there was used a sample havingsuch a concentration that its absorbance was 0.8 at pH 6.4 (the optimumfor TRACP 5b). As to a method for the assay, a plate having ananti-TRACP 5b antibody Trk27 immobilized thereon was used and 100 μl ofeach sample was added to each well and stirred with shaking at roomtemperature for 1 hour. Then, the reaction solution was discarded andeach well was washed three times with 200 μl of Tris buffer containing0.05% Tween 20. Thereafter, 100 μl of the substrate buffer solution wasadded to each well and shaken for 30 seconds, followed by incubation inan incubator at 37° C. for 1 hour. Then, 50 μl of 0.2N NaOH was added toeach well to terminate the enzyme reaction and the absorbance of thereaction solution was measured with a microtiter plate reader. Theresults are shown in FIG. 1. Under such conditions, the optimum pH forTRACP 5b was about 6.4 and that for TRACP 5a was 5.6.

EXAMPLE 2

Comparison Between the Present Invention and a Method Using PNPP (aConventional Substrate)

Absorbance values due to the reaction of a buffer solution containing asubstrate CNPP with each of TRACP 5a and TRACP 5b at pHs 6.2, 6.4, 6.6and 6.8 were compared with results obtained in a comparative exampleusing a buffer solution (pH 6.1) containing, as a substrate, PNPP thatis considered suitable for the assay of TRACP 5b. According to themethod disclosed in Japanese Patent Application Kohyo No. 2002-510050,the composition of this PNPP substrate buffer solution was as follows:PNPP 8 mM, sodium acetate 0.1M, sodium tartrate 40 mM, pH 6.1. As asample of TRACP 5b, the same sample as in Example 1 was used. As asample of TRACP 5a, a sample was used which had been prepared so that inthe comparative example (PNPP was used at pH 6.1), a value for TRACP 5amight be one-tenth that for TRACP 5b according to the data disclosed inJapanese Patent Application Kohyo No. 2002-510050. The same assayprocedure as in Example 1 was carried out and absorbance was used as anindication of the enzymatic activity. The results are shown in Table 1.As is clear from the results, the 5b/5a ratios at pHs 6.4 and 6.6 in thecase of CNPP are 17 and 18, respectively, indicating that the method ofthe present invention is more suitable for assay of TRACP 5b than theconventional method (the comparative example).

TABLE 1 Comparative Example 2 (CNPP) Example (PNPP) Sample pH 6.2 pH 6.4pH 6.6 pH 6.9 pH 6.1 TRACP 5a 0.062 0.047 0.038 0.025 0.098 TRACP 5b0.802 0.811 0.674 0.401 0.981 TRACP 5b/5a 13 17 18 16 10

EXAMPLE 3

Method for Assaying TRACP 5b Activity by a Rate Method

To each well of a plate having an anti-TRACP 5b antibody TrK27immobilized thereon was applied 100 μl of a specimen (a standardsolution or serum), and stirred with shaking at room temperature for 1hour. Then, the reaction solution was discarded and each well was washedthree times with 200 μL of Tris buffer containing 0.05% Tween 20.Thereafter, 100 μl of a buffer solution containing a substrate (CNPP)was added to each well and shaken for 30 seconds, and then absorbance at405 nm was measured with a microtiter plate reader. Subsequently, theplate was heated in an incubator at 37° C. for 60 minutes. During theincubation, absorbance at 405 nm was measured at intervals of 10minutes. On the other hand, the following method was adopted as acomparative method (an end-point method): 60 minutes after the start ofthe incubation, 50 μl of 0.2N NaOH was added to each well as areaction-terminating solution and absorbance at 405 nm was similarlymeasured. FIG. 2 shows the reaction time course studied over a period of60 minutes. The TRACP 5b activity level is calculated by the followingequation:U/L=ΔOD/min of specimen/ΔOD/min of standard solution×activity value ofstandard solution wherein ΔOD/min is an absorbance change per minute ata measuring wavelength of 405 nm.

A rate method was practiced by measuring an absorbance change per minuteduring a period between 10 minutes and 20 minutes after the start of theincubation, and calculating the activity by the above equation. As aresult, the TRACP 5b activity levels of serum specimens 1 and 2 werefound to be 4.1 U/L and 9.1 U/L. These levels were substantially thesame as 4.2 U/L and 9.3 U/L which were levels measured after theaddition of the reaction-terminating solution after 60 minutes of thereaction in the end-point method. Therefore, it has been proved that thepresent invention makes it possible to assay TRACP 5b activity in about20 minutes by the rate method without using a terminating solution.

INDUSTRIAL APPLICABILITY

As explained above in detail, the immunoassay method of the presentinvention makes it possible to assay TRACP 5b in a specimen specificallywith high sensitivity with almost no influence of TRACP 5a in thespecimen. Therefore, TRACP 5b can be more accurately assayed thanbefore.

1. An immunoassay method for determining tartrate resistant acidphosphatase 5b (TRACP 5b) in a specimen, the method comprises bindingboth TRACP 5b and TRACP 5a in the specimen to an antibody that isreactive with both of TRACP 5b and TRACP 5a, separating the TRACP 5a andTRACP 5b bound to the antibody, and subjecting the TRACP 5a and TRACP 5bbound to the antibody to enzyme reaction with a2-halo-4-nitrophenylphosphoric acid represented by the general formula(1):

wherein X is Cl, or a salt of the compound of general formula (I), whichis a substrate more specific for TRACP 5b than for TRACP 5a, and thenassaying the enzymatic activity of said enzyme reaction to determineTRACP 5b to carry out the immunoassay of TRACP 5b in the specimen,wherein at the time of the enzyme reaction, the pH is higher than 6.3and not higher than 6.8.
 2. An immunoassay method according to claim 1,wherein at the time of the enzyme reaction, the pH is 6.35 to 6.75. 3.An immunoassay method according to claim 2, wherein the assay of theenzymatic activity is carried out by an end-point method.
 4. Animmunoassay method according to claim 1, wherein the assay of theenzymatic activity is carried out by an end-point method.